AN IMPROVED CA-45 PROTOCOL FOR INVESTIGATING PHYSIOLOGICAL-MECHANISMS IN CORAL CALCIFICATION

A sensitive experimental protocol using cloned corals (hereafter ''microcolonies'') of the branching scleractinian coral Stylophora pistillata and Ca-45 has been developed to enable reproducible measurements of physiological and biochemical mechanisms involved in calcium transport and compartmentalization during coral calcification. Cloned S. pistillata microcolonies were propagated in the laboratory from small fragments of parent colonies collected in 1990 in the Gulf of Aqaba, Jordan. Cloned microcolonies have several intrinsic properties that help to reduce unwanted biological variability: (1) same genotype; (2) similar sizes and shapes; and (3) absence of macroscopic boring organisms. Errors specifically associated with long-standing problems to do with isotopic exchange were further reduced by producing microcolonies with no skeletal surfaces exposed to the radioisotope-labelled incubation medium. The value of the technique resides principally in its superior ability to elucidate transportation pathways and processes and not in its ability to quantitatively estimate calcium deposition by corals in nature. We describe here a rapidly exchangeable calcium pool in which up to 90% of the radioactive label taken up during incubations is located. This pool (72.9 +/- 1.4 nmol Ca mg-l protein) is presumably located within the coelenteric cavity as suggested by the following: (1) it has 4-min halftime saturation kinetics; (2) the accumulation of calcium is linearly correlated with the calcium concentration of seawater; and (3) its insensitivity to metabolic and ion transport inhibitors indicate that membranes do not isolate this compartment. Washout of this large extracellular pool greatly improved estimates of calcium deposition as evidenced by 10 to 40% reduction in coefficients of variation when compared with previous Ca-45(2+) methods described in the literature. Comparisons of calcification measurements simultaneously carried out using the alkalinity anomaly technique and the Ca-45 protocol described here show that the correlation coefficient of both techniques is close to 1. Unlike previous reports, our Ca-45(2+)-derived measurements are slightly lower than those computed from the alkalinity depletion technique.